US20200392468A1 - Addition of nucleases directly to cell culture to facilitate digestion and clearance of host cell nucleic acids - Google Patents

Addition of nucleases directly to cell culture to facilitate digestion and clearance of host cell nucleic acids Download PDF

Info

Publication number
US20200392468A1
US20200392468A1 US16/498,831 US201816498831A US2020392468A1 US 20200392468 A1 US20200392468 A1 US 20200392468A1 US 201816498831 A US201816498831 A US 201816498831A US 2020392468 A1 US2020392468 A1 US 2020392468A1
Authority
US
United States
Prior art keywords
virus
cells
benzonase
endonuclease
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US16/498,831
Other languages
English (en)
Inventor
Yi Li
Matthew Woodling
Adam Kristopeit
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck Sharp and Dohme LLC
Original Assignee
Merck Sharp and Dohme LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck Sharp and Dohme LLC filed Critical Merck Sharp and Dohme LLC
Priority to US16/498,831 priority Critical patent/US20200392468A1/en
Publication of US20200392468A1 publication Critical patent/US20200392468A1/en
Assigned to MERCK SHARP & DOHME CORP reassignment MERCK SHARP & DOHME CORP ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LI, YI, KRISTOPEIT, Adam, WOODLING, MATTHEW
Assigned to MERCK SHARP & DOHME LLC reassignment MERCK SHARP & DOHME LLC MERGER (SEE DOCUMENT FOR DETAILS). Assignors: MERCK SHARP & DOHME CORP.
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K4/00—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
    • C07K4/04—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/73—Hydrolases (EC 3.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2531/00—Microcarriers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/10—Mineral substrates
    • C12N2533/12—Glass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/78—Cellulose
    • C12N2700/00—
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16151—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C12N2710/16161—Methods of inactivation or attenuation
    • C12N2710/16162—Methods of inactivation or attenuation by genetic engineering
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24151—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2796/00—Viruses not covered by groups C12N2710/00 - C12N2795/00

Definitions

  • the present invention is in the field of virology and vaccine development and relates to an improved method of manufacture of a viral vaccine, particularly of a whole-virus vaccine, preferably of an attenuated live vaccine.
  • HcDNA host cell DNA
  • cGMP Current Good Manufacturing Practices
  • HcDNA/HcRNA The reduction or removal of HcDNA/HcRNA during downstream processing is often facilitated by a dedicated enzymatic nucleic acid digestion step and/or purification steps (e.g., chromatography) to decrease the amount of nucleic acids and/or their size.
  • a dedicated enzymatic nucleic acid digestion step and/or purification steps e.g., chromatography
  • a live virus vaccine or viral vector is produced by infection of mammalian host cells grown in a bioreactor vessel. Following cell separation, filtered culture medium containing the crude vaccine product is incubated with an endonuclease, such as Benzonase® for a defined period of time to allow digestion of HcDNA/HcRNA after which the nucleic acid fragments are removed by one of various unit operations including diafiltration, chromatography, centrifugation, etc. See, e.g., Li et al., 2014, Biologicals 42(5):271-6; Puig et al., 2014, Methods Mol Biol. 1089:197-210; Langfield et al., 2011, Methods Mol Biol.
  • European Patent Application Publication EP 0870508A1 discloses a method to produce a viral antigen vaccine by infecting an animal cell line with virus, propagating virus in the cell culture in a fermenter, adding a nuclease to the cell culture shortly before the end of virus propagation to digest nucleic acid material released from the lysing host cells into the medium, harvesting the virus and obtaining viral antigens thereof by extraction in order to make the viral antigen vaccine.
  • European Patent No. EP 1358319B1 discloses a method to produce a live attenuated influenza vaccine in a single step procedure involving additional of an endonuclease, such as Benzonase®, and a protease to the virus-infected cell suspension culture in Roux bottles or roller bottles that does not require any chromatographic or other purification steps of the virus suspension harvested from the cell culture supernatant by centrifugation, particularly no protein separation or purification steps.
  • an endonuclease such as Benzonase®
  • protease to the virus-infected cell suspension culture in Roux bottles or roller bottles that does not require any chromatographic or other purification steps of the virus suspension harvested from the cell culture supernatant by centrifugation, particularly no protein separation or purification steps.
  • the present invention relates to the addition of an endonuclease (e.g., Benzonase®) to an infected cell culture to realize efficient HcDNA removal.
  • an endonuclease e.g., Benzonase®
  • the present invention relates to a method for making a vaccine, comprising the steps of: a) culturing ARPE-19 cells or Vero cells, as adherent cells on static surfaces or on microcarriers, b) infecting the ARPE-19 cells with cytomegalovirus or the Vero cells with a flavivirus (e.g., a Dengue virus); and c) treating the infected cell supernatant with Benzonase® and its co-factor, magnesium, during the virus production phase (post- or at infection prior to supernatant harvest), as to digest free HcDNA from lysed cells.
  • the cells are ARPE-19 cells cultured on microcarriers.
  • the cells are Vero cells cultured on
  • FIG. 1 is a plot of ARPE-19 cell viability on microcarriers (as measured by a NucleoCounter® cell counter) over time before supernatant harvest with different concentrations of Benzonase®.
  • FIGS. 2A-D are plots of various indicators of ARPE-19 cell metabolism grown adherently on microcarriers in the presence of Benzonase® (A: glucose; B: lactate; C: glutamine; D: pH) as measured by a BioProfile® Flex over time before supernatant harvest.
  • Benzonase® A: glucose; B: lactate; C: glutamine; D: pH
  • FIGS. 3A-C are plots of various indicators of HCMV virus production (A: HCMV-specific ELISA; B: qPCR for viral genomes; C: Relative Infectivity) in ARPE-19 cells grown adherently on microcarriers over time before supernatant harvest.
  • A HCMV-specific ELISA
  • B qPCR for viral genomes
  • C Relative Infectivity
  • FIG. 4 is a plot of protein concentration in HCMV-infected ARPE-19 culture (as measured by the Bradford assay) grown adherently on microcarriers over time before supernatant harvest.
  • FIGS. 5A-B are plots of ARPE-19 host cell DNA in cell free supernatants (as measured by quantitative PCR) over time before supernatant harvest.
  • FIG. 5B is a zoomed in view of FIG. 5A .
  • FIG. 6 is a plot of DNA concentration in cell free supernatants (as measured by the PicoGreen assay) over time before supernatant harvest.
  • FIG. 7 is a plot of ARPE-19 host cell DNA in cell free supernatants (as measured by quantitative PCR) in HCMV spinner flasks post-harvest with varying concentrations of Benzonase® added either 8 days prior to supernatant harvest (experimental arms) or post harvest and post clarification for 2 hrs (control).
  • FIG. 8 is a plot of ARPE-19 host cell DNA in cell free supernatants (as measured by quantitative PCR) in HCMV infected spinner flasks post-harvest with varying concentrations of Benzonase® added either 8 days prior to supernatant harvest (experimental arms) or post harvest and post clarification for 2 hrs (control) normalized to virus dose of 100 units.
  • FIGS. 9A-B are plots of ARPE-19 host cell DNA in cell free supernatants (as measured by quantitative PCR) in 3L reactor pairs on the day before harvest just prior to Benzonase® addition (A: reactor conditions 1-4; B: reactor conditions 5-8).
  • FIGS. 10A-B are plots of ARPE-19 host cell DNA in cell free supernatants (as measured by quantitative PCR) in 3L reactor pairs post harvest and post clarification, and in the case of the control arms, post 2 hr Benzonase® incubation after clarification (A: reactor conditions 1-4, 80 U/ml; B: reactor conditions 5-8, 20 U/ml).
  • FIGS. 11A-B are plots of HCMV viral mass (A: as measured by Western HCMV glycoprotein assay) and viral infectivity (B: as measured by cell based infectivity assay) under various reactor conditions post harvest and post clarification, and in the case of the control arms, 2 hr post Benzonase® incubation after clarification.
  • FIG. 12 Vero HcDNA in cell free supernatants as measured by qPCR on process samples from Dengue infected Vero harvests. Samples pulled after post harvest and post filtration overnight incubation. Error bars indicate standard deviation of three results, one from each flask, as each arm was run in triplicate.
  • FIGS. 13A-B Dengue RNA genome qPCR and Dengue virus relative potency on samples pulled after overnight incubation (post harvest and post filtration). Error bars indicate standard deviation of three results, one from each flask, as each arm was run in triplicate.
  • the present invention relates to the addition of a nuclease, such as Benzonase®, directly to a bioreactor or cell culture flask in which cell culture and viral vaccine production occur.
  • a nuclease such as Benzonase®
  • the present invention is based on simplification of the bioprocess for vaccine manufacturing.
  • the present invention is based on the addition of a single dose of highly active endonuclease.
  • HCMV human cytomegalovirus
  • ARPE-19 infected human retinal pigment epithelial cells
  • Vero live attenuated Dengue vaccine produced from infected monkey kidney epithelial cells (Vero) grown as adherent cells on static cell culture surfaces such as flasks or grown adherently on microcarriers, and is expected to be broadly applicable to other mammalian cell substrates used to produce viral vaccines.
  • nuclease directly to cell culture was demonstrated to be non-detrimental to ARPE19 or Vero cell culture as well as human cytomegalovirus (HCMV) or Dengue virus production in their respective cell lines. Moreover, the addition of nuclease was specifically demonstrated for a HCMV virus infected ARPE-19 cell culture in a bioreactor platform with microcarriers. In particular, it was unexpected that this approach would work with cells grown adherently on microcarriers. Growing cells on microcarriers, compared to adherent cells or suspensions in roller bottles, requires greater agitation to suspend the microcarriers and subjects the cells to greater shear stress.
  • nuclease post-harvest (1) purification processing time is reduced by elimination of a separate nucleic acid digestion step which generally increases viral potency yield; (2) the efficiency of nucleic acid digestion is increased since digestion in the bioreactor occurs for 0.5-8.0 days under cell culture conditions (during virus production) as opposed to a separate stand-alone unit operation carried out in a holding tank typically for 1-12 hr at room temperature or at 2-8° C.
  • nuclease e.g., Benzonase®
  • Benzonase® an endonuclease
  • viral stability was observed over longer incubation times with the endonuclease (up to 8 days for HCMV or up to 15 days for Dengue).
  • the present invention is directed to a method for production of a virus, comprising the steps of: a) infecting a culture of cells in a cell culture medium with a desired virus; and b) incubating the cells in cell culture medium containing endonuclease for up to 8 days for HCMV and up to 15 days for Dengue, wherein the endonuclease is added to the cell culture medium at any time from initial infection of the cells to prior to harvesting the cells.
  • the cells are grown adherently on microcarriers.
  • the cells are adhered to a static plastic surface. Examples of static plastic surfaces include, but are not limited to, cell stacks, cell factory systems, T-flasks, HYPERFlasksTM, HYPERStacksTM, and the like.
  • the endonuclease is added at the time of initial infection. In certain embodiments, the endonuclease is added from 0.5 to 8 days prior to harvesting the cells for HCMV or from 0.5 to 15 days prior to harvesting the cells for Dengue. Included herein is adding the endonuclease from 0.5, 1, 2, 3, 4, 5, 6, 7, 8, days prior to harvesting the cells for HCMV or Dengue, and additional from 9, 10, 11, 12, 13, 14, or 15 days prior to harvesting the Dengue cells. In certain embodiments, the endonuclease can be added multiple times during the virus production phase. For example, the endonuclease can be added 2, 3, 4, 5, 6, 7, 8, 9 or 10 times during the virus production phase.
  • the total amount of endonuclease added can be equivalent to the amounts used for a single addition as described herein or the total amount of endonuclease can be a multiple of the amount for a single addition (e.g., 2 ⁇ , 3 ⁇ , 4 ⁇ , 5 ⁇ , 6 ⁇ , 7 ⁇ , 8 ⁇ , 9 ⁇ , or 10 ⁇ the amount of a single addition as described herein).
  • the endonuclease can be continuously added, for example, through perfusion, using overall amounts of endonuclease as described herein.
  • the cells used for cell culture and viral propagation may be primary cells or any cultured cell line suitable for producing the virus.
  • the cells are obtained from a continuous cell line.
  • examples of cells which may be used include mammalian cells (e.g., ARPE-19, CHO, BHK, African green monkey kidney derived Vero, Madin-Darby Canine Kidney cells (MDCK), PBS-1 cells, HeLa cells, RK, RK44, RK13, MRC-5, CEF, diploid monolayer cells or PER.C6® cells), avian cells (e.g, chicken embryo fibroblasts, or continuous cell lines from an avian) and insect cells (e.g, Sf9 cells).
  • mammalian cells e.g., ARPE-19, CHO, BHK, African green monkey kidney derived Vero, Madin-Darby Canine Kidney cells (MDCK), PBS-1 cells, HeLa cells, RK, RK44, RK13, MRC-5, CEF, diploid monolayer cells or
  • the cells may be grown adherently on static plastic surfaces, such as in roller bottles, flasks, cell factories, or may be grown adherently on microcarriers, or in suspension cultures in stirred-tank bioreactors or disposable wave bioreactors.
  • the microcarriers may be made of any suitable surface for cell culture, including, but not limited to, dextran, collagen, polystyrene, polyacrylamide, gelatine, glass, cellulose, polyethylene and plastic.
  • the cells are grown adherently on microcarriers.
  • the microcarriers are Cytodex® 1 microcarriers, dextran-based spheres with diethylaminoethyl functionality, available from GE Healthcare Life Sciences.
  • the cells are in form of a cell culture.
  • Cell culture media used in the methods of the invention can be selected from standard media, sometimes with serum/other supplements, or with media specifically developed for the application.
  • the cell culture media contains serum.
  • the cell culture medium in which the infected cells are grown contains no, or very minimal, amounts of trypsin.
  • trypsin is not added to the cell culture medium in which the infected cells are grown.
  • the viruses are selected from enveloped DNA or RNA viruses, with single or double (DNA) stranded genomes, sense or antisense, continuous or segmented.
  • the viruses are selected from the group of enveloped viruses, including, flaviviruses, togaviruses, retroviruses, coronaviruses, filoviruses, rhabdoviruses, bunyaviruses, orthomyxoviruses, paramyxoviruses, arenaviruses, hepadnaviruses, herpesviruses, and poxviruses.
  • the viruses are flaviruses, coronaviruses, orthomyxoviruses, herpesviruses or togaviruses.
  • the virus is selected from the herpesviruses, for example, a cytomegalovirus strain or a flavivirus, for example, a Dengue virus.
  • the cytomegalovirus is a genetically attenuated human cytomegalovirus as described in U.S. Pat. No. 9,546,355, the contents of which are incorporated by reference in its entirety.
  • the flavivirus is a chimeric flavivirus, wherein the viral genome comprises a backbone of a first flavivirus (including C, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 genes) and the preMembrane (prM) and envelope (E) genes of a second flavivirus, wherein the second flavivirus is selected from DENV1, DENV2, DENV3 or DENV4.
  • the first flavivirus can be a different dengue serotype or another flavivirus, such as yellow fever virus.
  • the flavivirus is a Dengue virus.
  • the Dengue virus is a dengue virus from one of the 4 dengue virus serotypes, referred herein as DEN1 (dengue virus serotype 1), DEN2 (dengue virus serotype 2), DEN3 (dengue virus serotype 3), or DEN4 (dengue virus serotype 4).
  • the Dengue virus comprises a viral genome that comprises a TL-2 ⁇ 30 modification in the 3′UTR.
  • the Dengue virus is a DEN1 virus wherein the viral genome of DEN1 comprises a 30 nt deletion corresponding to the TL2 stem-loop structure in the 3′ UTR (rDEN1 ⁇ 30).
  • the dengue virus is a DEN2 virus comprising the DEN2 prM and E genes on a DEN4 backbone, wherein the DEN4 backbone comprises a 30-nt deletion corresponding to the TL2 stem-loop structure in the 3′ UTR (rDEN2/4 ⁇ 30).
  • the Dengue virus is a DEN3 virus wherein the DEN3 viral genome comprises a 30 nt deletion corresponding to the TL2 stem-loop structure in the 3′ UTR and a separate, noncontiguous, upstream 31 nucleotide deletion corresponding to the TL-3 structure of the 3′ UTR (rDEN3 ⁇ 30/ ⁇ 31).
  • the Dengue virus is a DEN4 virus, wherein the DEN4 viral genome comprises a 30 nucleotide deletion corresponding to the TL2 stem-loop structure in the 3′ UTR (rDEN4 ⁇ 30).
  • the virus may be a primary viral isolate directly obtained from an infected individual, a genetically engineered attenuated virus, a genetically-engineered replication-deficient virus, a cell line passaged adapted virus, a cold-adapted virus, a temperature-sensitive mutant virus, or a genetically engineered re-assortant virus.
  • Endonucleases can be classified based on their substrates as follows: deoxyribonucleases (DNases) which degrade DNA, i.e., have DNA specificity; ribonucleases (RNases) which degrade RNA, i.e., have RNA specificity; and endonucleases that degrade DNA and RNA, i.e., have specificity for both DNA and RNA.
  • DNases include but are not limited to DNase I, DNase II and endodeoxyribonuclease IV.
  • RNases include but are not limited to RNase I, RNase III, RNAse E, RNAse F and RNAse P.
  • RNAse E RNAse E
  • RNAse F RNAse P
  • Benzonase® a broad spectrum endonuclease derived from the bacterium Serratia marcescens . See, e.g., Eaves et al., 1963, J. Bacteriol. 85:273-278.
  • Benzonase® is a genetically engineered endonuclease which degrades both DNA and RNA strands in many forms by hydrolyzing internal phosphodiester bonds between specific nucleotides to form 5′-monophosphate terminated oligonucleotides which are 3 to 8 bases in length. It promotes quick reduction of the viscosity of cell lysates, which facilitates ultracentrifugation. It reduces proteolysis and increases the yield in targeted protein and offers complete elimination of nucleic acids from, e.g. recombinant, proteins. It has an exceptionally high activity of 400,000 U/mg.
  • the endonuclease e.g. Benzonase®
  • the endonuclease is added once to the medium at a very low initial concentration of 5-500 units/ml, 10-250 units/ml or 20-80 units/ml (or as low as 2.5 U/mL; or 5 U/mL as exemplified for the Dengue vaccine) of medium.
  • the endonuclease effectively clears (to below 20 ng/ml in the supernatant) the cell culture medium of free DNA and/or RNA originating mainly from lysing or lysed host cells.
  • the amount of host cell DNA is less than 20 ng/ml as measured by qPCR.
  • the endonuclease is added at a concentration of 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 25, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450 or 500 units/ml of medium.
  • the use, under the same conditions, of 20-80 units/ml of Benzonase® leads to a superior decrease of DNA concentration when the endonuclease is added during cell culture compared with the use of 80 units/ml Benzonase® applied post harvest, with no impact to cell growth or virus production, surprisingly also when cells are grown adherently on microcarriers.
  • the virus is placed in contact with the cells grown adherently on microcarriers or T-flasks (HCMV in ARPE-19 or Dengue in Vero) to allow the virus to infect the cells and to propagate.
  • the viral seed lot is added to the cell culture and allowed to absorb on the cells, for instance, for about 30 minutes with gentle mixing (e.g., about 30 rpm), after which further culture medium may be added and the pH adjusted if desired. Stirring speed may be adjusted and the culture maintained. Following infection, amplification of the number of virus particles takes place.
  • This step can suitably be performed in bioreactors, for instance, at scales of between 1 and 20,000 liters, e.g., between 10 and 2,000 liters, e.g., between 50 and 1,000 liters, which scale can easily be adjusted to the demand for the vaccine.
  • the bioreactor is a single use bioreactor (SUB).
  • the reaction conditions within the bioreactor for activity of the endonuclease include 1) a pH between 6.0 and 9.0, preferably between 7.0 and 8.0, and more preferably a pH of 7.2; 2) a temperature between 25° C. and 45° C., preferably at a temperature comprised between 30° C. and 40° C., more preferably at a temperature comprised between 35° C. and 40° C., and even more preferably at a temperature of 37° C.; and 3) a cofactor such as MgCl 2 at a concentration of 2 mM.
  • MgCl 2 can be used at concentrations 1.0 mM to 10 mM, or 1 mM to 5 mM.
  • Other divalent cations such as CaCl 2 may be suitable for use.
  • the endonuclease is typically added to the culture media post infection, up to 8 days prior to supernatant harvest.
  • the endonuclease is added concurrently with virus infection or just after virus infection, e.g., within 1, 2, or 3 hours post infection.
  • the culture is permitted to proceed for up to 15 days prior to harvesting. In some embodiments where the Dengue virus is propagated, the culture is permitted to proceed for 7 days or up to 8 days prior to harvesting. In other embodiments where the Dengue virus is propagated the culture is permitted to proceed for up to 9, 10, 11, 12, 13, 14, or 15 days prior to harvesting.
  • reaction conditions within the HCMV infected ARPE-19 microcarrier bioreactor for efficient activity of endonuclease have been demonstrated at 1) a pH between 6.6-7.3, 2) an endonuclease concentration of 20-80 U/mL culture, 3) a MgCl 2 cofactor concentration of 2 mM, and 4) a temperature between 35° C. and 39° C.
  • Endonuclease addition time has been demonstrated post infection, from 0.5 to 8 days prior to virus product harvest, with more efficient digestion at earlier additions.
  • the reaction conditions within the Dengue-virus-infected Vero T-225 flasks have been demonstrated at 1) a pH between 6.8 and 7.3, 2) an endonuclease concentration of 5-20 U/mL culture, 3) a MgCl 2 cofactor concentration of 2 mM, and 4) a temperature of 37° C.
  • Endonuclease addition time has been demonstrated at infection, up to 7 days prior to virus product harvest. In certain embodiments, however, endonuclease addition will occur at infection up to 15 days prior to virus product harvest.
  • the reaction conditions are for Dengue virus infected Vero microcarrier bioreactors.
  • the virus or components thereof are harvested from the cell culture. This can be done by routine methods, which are known to the skilled person.
  • the virus produced and released in the cell culture medium can be separated from the cellular biomass by conventional methods, such as clarifying filtration, centrifugation, or ultrafiltration, and harvested in the supernatant.
  • Conventional processes for harvesting the virus can be used, for instance, those described in U.S. Pat. No. 4,525,349.
  • the liquid medium suspension containing the virus is typically withdrawn, filtered and concentrated by, for instance, ultrafiltration. For instance, at the end of the culture, harvesting is carried out by collecting the culture medium containing the viral suspension.
  • the clarified harvest can optionally be ultrafiltrated to concentrate the viral suspension, and subsequently, the virus can be purified using any of several protocols known to one skilled in the art, e.g., using gel filtration and/or ion exchange chromatography, for instance, following the procedures as described in U.S. Pat. No. 4,525,349, the contents of which are incorporated herein.
  • the resulting concentrated virus suspension can optionally be diluted, and subsequently inactivated if required, for which conventional methods can be used.
  • the virus is further processed to a split virus comprising any one of the following steps of dilution, homogenization, nuclease treatment, pressure filtration, ultra/diafiltration, solubilisation, diafiltration, chromatography, stabilization by formaldehyde treatment, dilution, ultra/diafiltration, (detergent) stabilizer addition, a second homogenisation and sterile filtration.
  • Clarification of the mixture obtained allows under suitable conditions the withdrawal of the cellular debris. Clarification is preferably performed by depth filtration.
  • Depth filtration includes but is not limited to the use of one or more commercially available products such as Sartopure® filters from Sartorius (e.g. Sartopure®), CUNO Incorporated AP series depth filters (e.g. AP01), CUNO Incorporated CP series depth filters (e.g. CP10, CP30, CP50, CP60, CP70, CP90), CUNO Incorporated HP series depth filters (e.g. HP10, HP30, HP50, HP60, HP70, HP90), CUNO Incorporated Calif series depth filters (e.g.
  • concentration step is performed by ultrafiltration.
  • the ultrafiltration is preferably a cross-flow filtration.
  • the principle of cross-flow filtration is known to the person skilled in the art (see, e.g., Richards, G. P. and Goldmintz, D., J. Virol. Methods (1982), 4 (3), pages 147-153. “Evaluation of a cross-flow filtration technique for extraction of polioviruses from inoculated oyster tissue homogenates”).
  • Diafiltration of the fraction is an improvement of microfiltration and involves diluting said fraction comprising the viruses with a solution to effect a reduction in the concentration of the impurities in said fraction.
  • the dilution of the fraction comprising the viruses allows washing out more of the impurities from said fraction.
  • the diafiltration may be carried out in a batch mode, semi-continuous mode, or a continuous mode.
  • the diafiltration step can be advantageously used to change the buffer in which the virus is comprised as well as selective remove impurities by size. It can be useful to exchange the buffer used in the purification process against a pharmaceutically acceptable buffer.
  • Filters used according to the invention are preferably amenable to sterile processing.
  • Autoclaveable filters are commercially available such as UFP hollow fiber membranes (GE Healthcare) or Prostak Microfiltration Modules (Millipore).
  • Gamma irradiated membranes are commercially available as well, such as RTP hollow fibers (GE Healthcare) or irradiated hollow fibers from Spectrum Labs. Diafiltration of the fraction comprising the viruses is preferably performed over filters having a pore size of ⁇ 0.1 ⁇ m.
  • chromatographic separations may be used to isolate the virus from impurities. Such separations can occur via virus bind and elute or virus flowthrough modes utilizing various types of separation, including but not exclusively, ion exchange, hydrophobic interaction, size exclusion, and multi modal ligands.
  • Preferred chromatographic platforms include those suited for large molecules, such as monolith columns or membrane absorbers, such as those commercially available from Sartorius (Sartobind®), Bia Separations (CIM monolithic columns), and Natrix Separations (NatrifloTM).
  • Resin based chromatographic platforms such as those offered by EMD Millipore (Eshmuno®, Fractogel®), GE Healthcare (CAPTO), and Tosoh Biosciences (Toyopearl®) may also be effectively used to isolate virus from impurities.
  • the whole-virus vaccines prepared using the methods of the present invention may be used for the prophylactic or therapeutic treatment of viral infections. They may be administered as known in the art, e.g. intravenously, subcutaneously, intramuscularly or intranasally.
  • the virus strains disclosed herein and the vaccines made thereof may, however, also be used as vectors or shuttles to present heterologous antigens to the immune system, e.g. antigens of viral envelope proteins such HIV-1 or hepatitis antigens.
  • a NucleoCounter® NC-200TM instrument (ChemoMetec Inc., Davis, Calif.) was used to measure culture cell count and viability. The instrument counted cells and cell viability of samples containing cells on microcarriers via a nuclear stain and cell lysis buffering system.
  • the sandwich enzyme linked immunosorbent assay used an anti-HCMV monoclonal antibody for capture and a polyclonal antibody for detection, with an alkaline phosphatase conjugated secondary antibody. Results are reported relative purified HCMV reference standard.
  • Infectious particles were measured via a cell-based assay using human retinal epithelial cells planted in 96-well or 384-well microtiter plates. Cells were incubated for 24 hours, and then infected with serial dilutions of HCMV samples or reference standard. The infected cells were incubated for 24 hours and fixed with a dilute formaldehyde solution.
  • a dose response curve was generated from the % infection signal to calculate the potency of each sample relative to the reference standard.
  • HCMV genome concentration was measured by a real-time quantitative PCR standard curve assay (standard curve range: 1E9 copies/mL-1E5 copies/mL).
  • the PierceTM 660 nm Assay (ThermoFisher Scientific Inc., Waltham, Mass.) was used to quantify total protein concentration in vaccine process intermediates and final product samples.
  • a standard curve was prepared from Bovine Serum Albumin (BSA), and samples were diluted and incubated in the presence of Benzonase® to reduce nucleic acid interference.
  • the PierceTM 660 nm Assay Reagent was added, the plate was incubated at room temperature and the absorbance was read at 660 nm. Unknown sample concentrations were determined by interpolation from the standard curve using a linear fit.
  • Total nucleic acids were isolated from the sample by proteinase K treatment, phenol-chloroform extraction, and alcohol precipitation. Host cell DNA concentration was measured by a real-time quantitative PCR standard curve assay (standard curve range: 200 ng/mL-20 pg/mL).
  • Residual DNA in process samples was quantified using the Quant-iTTM PicoGreen® dsDNA kit from Life Technologies-InvitrogenTM.
  • the Quant-iTTM PicoGreen® dsDNA reagent is a fluorescent dye that selectively binds double-stranded DNA.
  • a standard curve was prepared from the supplied ⁇ DNA standard, samples are diluted, the PicoGreen® reagent was added, and the fluorescence was measured at excitation 485 nm, emission 535 nm.
  • Quantitation of the DNase activity of Benzonase® in process samples was done using Amplifluor® UniPrimer II (EMD Millipore Corporation, Billerica, Mass.), a DNA hairpin substrate.
  • Amplifluor® UniPrimer II EMD Millipore Corporation, Billerica, Mass.
  • a standard curve was prepared from Benzonase®, samples were diluted, the substrate was added, the plate was incubated at 37° C. for 60 ⁇ 5 min, and the fluorescence was measured at excitation 485 nm, emission 535 nm.
  • the entire assay has been automated on a workstation (Tecan Systems Inc., San Jose, Calif.).
  • a Capillary Electrophoresis based Simple WesternTM was used to monitor virus mass.
  • the HCMV sample was reduced and denatured, then loaded to a capillary and separated by size, bound to the capillary using a photo-linking technology and probed with a rabbit HCMV glycoprotein monoclonal antibody.
  • a goat anti-rabbit HRP conjugate secondary antibody, luminol, and peroxide were utilized for chemiluminescence detection of the HCMV glycoprotein.
  • Virus concentration of test samples was determined relative to a purified HCMV reference standard of known virus concentration.
  • a quantitative polymerase chain reaction (qPCR) assay was used to quantify residual Vero cell DNA in samples.
  • the residual DNA method measures residual Vero DNA using a qPCR primer-probe set specific for the target host DNA sequence.
  • a test sample was pre-diluted as needed, and DNA was extracted from the test sample. The quantities of residual host cell DNA in the test sample were measured by interpolation against a Vero DNA standard curve.
  • the assay procedure was used to quantify serotype-specific virus genomes in dengue live attenuated virus vaccine samples.
  • the assay employs reverse transcription quantitative PCR (RT-QPCR) with Dengue virus serotype-specific QPCR primers/probe sets for detection.
  • Serotype-specific virus genome copy numbers in the test articles were determined by interpolation against standard curves of serotype-specific RNA transcripts.
  • Infectivity was measured via a cell-based assay using Vero (African Green Monkey kidney) cells planted in 96-well microtiter plates. Cells were incubated for 24 hours, and then infected with serial dilutions of Dengue samples or reference standard. The infected cells were incubated and then fixed with a dilute formaldehyde solution. Following fixing, cells were permeabilized using a 1% Triton X-100 solution and then incubated with the primary antibody [rabbit anti-DEN mAb] specific for the serotype being tested at 2-8° C. overnight on a rotating platform.
  • a conjugated secondary antibody [Goat anti-mouse IgG (H+L) IRDye 800CW—LI-COR] was added to the cells and finally the plates were dried and read on an OdysseyTM Automated Infrared Imaging System (Li-Cor Biosciences, Lincoln, Iowa). Determination of relative potency of each sample (relative to Dengue reference standard) using a 4-parameter fit parallel-line analysis was performed using SoftMax Pro (Molecular Devices).
  • ARPE-19 cells (ATCC No. CRL-2302, American Type Culture Collection, Manassas, Va.) were initially cultured in static vessels (i.e., T-225 Flask). Through cell expansion, cells were eventually cultured on microcarriers in a SOL stirred tank bioreactor. Upon appropriate cell growth in the SOL reactor, the cell culture was infected with HCMV. During virus production, material from the SOL microcarrier culture was sampled and dispensed into 125 mL disposable spinner flasks (Corning, Cat. No. 3152). Throughout the following operations, the spinner cultures were maintained in an incubator at 37° C. and 5% CO 2 under stirred base agitation, with exception to sampling and operations post harvest.
  • Benzonase® and MgCl 2 were added to three of the four spinner flasks (Arms A 1 -A 3 ). Specifically, a stock Benzonase® solution consisting of Benzonase® endonuclease (EMD Millipore, Billerica, Mass., Cat. No. 1.01697.0010) diluted in culture media to 12,500 Units Benzonase® (U)/mL solution was added to the three spinner flask arms, A 1 , A 2 , and A 3 to bring the culture concentration to 20, 40, and 80 U/mL Benzonase® respectively.
  • EMD Millipore Billerica, Mass., Cat. No. 1.01697.0010
  • U Benzonase®
  • HCMV virus production was analyzed over the course of the spinner cultures via HCMV ELISA, Infectivity, and qPCR for viral genomes. All three measures trended similarly over all four arms, indicating virus production and stability was not impacted by the presence of 20, 40, or 80 U/mL Benzonase® ( FIGS. 3A-3C ). Similarly, overall protein level in the culture supernatant was not impacted; indicating overall supernatant protein content was not impacted by the presence of 20, 40, or 80 U/mL Benzonase® ( FIG. 4 ).
  • all spinners containing Benzonase® contained >10 fold less host cell DNA than the non Benzonase® spinner.
  • the host cell DNA content of the clarified harvests from the three spinners containing Benzonase® was >1.9 fold less than that of the Benzonase® treated clarified harvest from the spinner not containing Benzonase® during culture (Arm C) ( FIG. 7 ). This indicates that equivalent or better DNA digestion can be achieved by adding Benzonase® to the culture as compared to a post-harvest digestion (2 hr Room Temp, 80 U/mL), even if the digestion post harvest contained up to 4 ⁇ more Benzonase® than that used in the culture.
  • ARPE-19 cells were expanded and planted into sixteen 3L reactor systems containing microcarriers through a process similar to that described in Example 1. After appropriate cell growth in the 3L reactors, the reactors were infected with HCMV. In this experiment, a pairwise comparison of Benzonase® addition during the culture or post harvest was made under eight different experimental conditions that varied with respect to cell culture conditions.
  • Benzonase® solution consisting of Benzonase® endonuclease diluted in culture media to 15,000 Units Benzonase® (U)/mL solution was added to eight of the 3L reactor systems, with four at 80 U/mL Benzonase® and four at 20 U/mL Benzonase®.
  • 0.1M MgCl 2 stock solution was added to all eight reactors to bring the culture concentration to 2 mM MgCl 2 .
  • the samples from tanks that had previously had no Benzonase® added were treated with 80 U/mL Benzonase® and 2 mM MgCl 2 for 2 hrs at room temperature.
  • Benzonase® endonuclease at 250,000 Units Benzonase® (U)/mL and 1 M MgCl 2 (Merck BSO, Cat 17RCM909) were added separately to achieve the desired concentrations.
  • the Benzonase® treated material was sampled for analysis.
  • On demand assays included measurements of cell count and viability via nucleo staining (Nucleocounter®) and supernatant metabolites via Bioprofile® (Nova Biomedical). Prior to freezing any sample, microcarriers were removed via a sieve, and whole cells were removed via syringe filtration (Pall Corporation, Cat. No. 4190). In addition, 60% (w/v) sucrose was added to the sample as a cryoprotectant to a final concentration of 10% sucrose.
  • the clarified sample on harvest day from the four reactors that received 20 U/mL Benzonase® 24 hr prior to harvest had similar HcDNA content to the associated paired reactors that had been treated post harvest, i.e., clarified and Benzonase® treated at 80 U/mL for 2 hrs at room temp.
  • the HcDNA content was ⁇ 10 ng/dose in all four reactors that received 80 U/mL Benzonase® 24 hrs prior to harvest. The same was not true for the 20 U/mL-treated and untreated bioreactors) ( FIGS. 10A-B ).
  • Vero cells were cultured on T-225 flasks and expanded until 14 flasks of confluent cells were attained. Upon confluence, the flasks were split into four different arms. Experimental arms included the addition of Benzonase® (added to the infection media containing Dengue), at concentrations of 5, 10, or 20 U/mL. The control arm was not treated with Benzonase®. The four arms (control, 5, 10, 20 U/mL Benzonase®) were run in triplicate, for a total of 12 flasks. The two remaining flasks of the original 14 were sacrificed at this point to attain a cell count just prior to infection.
  • Benzonase® added to the infection media containing Dengue
  • the control arm was not treated with Benzonase®.
  • the four arms were run in triplicate, for a total of 12 flasks. The two remaining flasks of the original 14 were sacrificed at this point to attain a cell count just prior to infection.
  • HcDNA clearance was established when adding Benzonase® at infection as opposed to post-harvest. Specifically, a lower amount of HcDNA per vaccine dose can be achieved per amount of Benzonase® addition when performed at infection as opposed to when performing as a post-harvest unit operation, allowing for the realization of process cost savings through reduction in the amount of Benzonase® needed.
  • Vero cells are expanded and planted into two 3L reactor systems containing microcarriers through a process similar to that described in Example 1. After appropriate cell growth in the 3L reactors, the reactors are infected with Dengue serotype 4 virus. In this experiment, a comparison of Benzonase® addition during the culture or post-harvest is made.
  • In-culture Benzonase® treatment is tested against the post-harvest Benzonase® condition for the respective duplicate reactor. Accordingly, two reactors are treated with 20 U/mL Benzonase® one day prior to harvest. More specifically, a stock Benzonase® solution consisting of Benzonase® endonuclease is diluted in 50-100 mL culture media and added to two of the 3L reactor systems to result in a final Benzonase® concentration of 20 units Benzonase® per mL cell culture. Following Benzonase® incubation the virus is harvested into a collection vessel and sampled for analysis. Post-harvest the virus is purified using the 1 day purification process detailed below
  • Virus from the control reactor that had previously had no Benzonase® added is harvested and treated with 20 U/mL Benzonase® for overnight at 2-8° C. Specifically, Benzonase® endonuclease at 250,000 Units Benzonase® (U)/mL is added to the harvested virus to result in a final concentration of 20 units Benzonase® per mL. Post addition the control arm is purified using the 2 day purification process detailed below.
  • On harvest day virus is sampled and analyzed via on demand assays or aliquoted and frozen for later analytical characterization.
  • On demand assays included measurements of cell count and viability via nucleo staining (Nucleocounter®) and supernatant metabolites via Bioprofile® (Nova Biomedical). Prior to freezing any sample, microcarriers are removed via a sieve.
  • Harvested virus that had been incubated with Benzonase® in the 3 L reactor are clarified by filtration using an Express SHC 0.5/0.2 um bilayer filter.
  • the virus is concentrated (5 ⁇ volume reduction) by means of tangential flow ultrafiltration across a 300 KDa nominal molecular weight cut-off flat-sheet membrane and diafiltered against 10 diavolumes of formulation buffer (11 mM Potassium Phosphate, 9% w/v sucrose, pH 7.5).
  • formulation buffer 11 mM Potassium Phosphate, 9% w/v sucrose, pH 7.5
  • the virus is sterile filtered using an Express SHC 0.5/0.2 um bilayer filter. Throughout purification process intermediates are maintained at 2-8° C.
  • Harvested virus from the control reactor is clarified by filtration using an Express SHC 0.5/0.2 um bilayer filter, immediately following Benzonase® addition.
  • the filtered virus is incubated at 2-8° C. overnight.
  • the virus is then concentrated (5 ⁇ volume reduction) by means of tangential flow ultrafiltration across a 300 KDa nominal molecular weight cut-off flat-sheet membrane and diafiltered against 10 diavolumes of formulation buffer (11 mM Potassium Phosphate, 9% w/v sucrose, pH 7.5).
  • formulation buffer 11 mM Potassium Phosphate, 9% w/v sucrose, pH 7.5
  • HV Harvested virus
  • FV filtered virus
  • UCR ultrafiltration concentrated retentate
  • UFFR ultrafiltration final retentate
  • SFP sterile filtered product

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
US16/498,831 2017-03-30 2018-03-28 Addition of nucleases directly to cell culture to facilitate digestion and clearance of host cell nucleic acids Abandoned US20200392468A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/498,831 US20200392468A1 (en) 2017-03-30 2018-03-28 Addition of nucleases directly to cell culture to facilitate digestion and clearance of host cell nucleic acids

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201762478895P 2017-03-30 2017-03-30
US16/498,831 US20200392468A1 (en) 2017-03-30 2018-03-28 Addition of nucleases directly to cell culture to facilitate digestion and clearance of host cell nucleic acids
PCT/US2018/024734 WO2018183426A1 (fr) 2017-03-30 2018-03-28 Addition de nucléases directement à une culture cellulaire pour faciliter la digestion et la clairance d'acides nucléiques de cellules hôtes

Publications (1)

Publication Number Publication Date
US20200392468A1 true US20200392468A1 (en) 2020-12-17

Family

ID=63676777

Family Applications (2)

Application Number Title Priority Date Filing Date
US16/498,831 Abandoned US20200392468A1 (en) 2017-03-30 2018-03-28 Addition of nucleases directly to cell culture to facilitate digestion and clearance of host cell nucleic acids
US16/498,013 Active US11268071B2 (en) 2017-03-30 2018-03-28 Addition of nucleases directly to cell culture to facilitate digestion and clearance of host cell nucleic acids

Family Applications After (1)

Application Number Title Priority Date Filing Date
US16/498,013 Active US11268071B2 (en) 2017-03-30 2018-03-28 Addition of nucleases directly to cell culture to facilitate digestion and clearance of host cell nucleic acids

Country Status (3)

Country Link
US (2) US20200392468A1 (fr)
EP (2) EP3601544A4 (fr)
WO (2) WO2018183429A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11268071B2 (en) 2017-03-30 2022-03-08 Merck Sharp And Dohme Corp. Addition of nucleases directly to cell culture to facilitate digestion and clearance of host cell nucleic acids

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3122866B1 (fr) 2014-03-25 2019-11-20 Terumo BCT, Inc. Remplacement passif de milieu
WO2017004592A1 (fr) 2015-07-02 2017-01-05 Terumo Bct, Inc. Croissance cellulaire à l'aide de stimuli mécaniques
EP3464565A4 (fr) 2016-05-25 2020-01-01 Terumo BCT, Inc. Expansion cellulaire
US11104874B2 (en) 2016-06-07 2021-08-31 Terumo Bct, Inc. Coating a bioreactor
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
EP3656842A1 (fr) 2017-03-31 2020-05-27 Terumo BCT, Inc. Expansion de cellules
SG11202005251VA (en) 2017-12-07 2020-07-29 Merck Sharp & Dohme Formulations of dengue virus vaccine compositions
US20210047626A1 (en) * 2018-04-24 2021-02-18 Merck Sharp & Dohme Corp. Scalable chromatography process for purification of human cytomegalovirus
EP4222250A1 (fr) * 2020-10-02 2023-08-09 Genelux Corporation Production de virus à partir de cellules adhérentes dans un bioréacteur

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW570803B (en) 1997-04-09 2004-01-11 Duphar Int Res Influenza vaccine
ES2327103T3 (es) * 2000-09-25 2009-10-26 Polymun Scientific Immunbiologische Forschung Gmbh Vacuna viva de influenzavirus y procedimiento de fabricacion.
US7195905B2 (en) * 2001-12-10 2007-03-27 Baxter Healthcare S.A. Enveloped virus vaccine and method for production
US20080193477A1 (en) 2005-08-10 2008-08-14 Acambis Inc. Vaccination Against Dengue Virus Infection
US9439960B2 (en) * 2007-10-10 2016-09-13 The Trustees Of Princeton University Cytomegalovirus vaccines and methods of production
KR20110074563A (ko) 2008-09-24 2011-06-30 메디뮨 엘엘씨 바이러스의 정제 방법
EP3601544A4 (fr) 2017-03-30 2021-01-27 Merck Sharp & Dohme Corp. Addition de nucléases directement à une culture cellulaire pour faciliter la digestion et la clairance d'acides nucléiques de cellules hôtes

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11268071B2 (en) 2017-03-30 2022-03-08 Merck Sharp And Dohme Corp. Addition of nucleases directly to cell culture to facilitate digestion and clearance of host cell nucleic acids

Also Published As

Publication number Publication date
EP3601544A1 (fr) 2020-02-05
EP3601543A1 (fr) 2020-02-05
EP3601543A4 (fr) 2021-01-27
US11268071B2 (en) 2022-03-08
WO2018183426A1 (fr) 2018-10-04
EP3601544A4 (fr) 2021-01-27
US20200040310A1 (en) 2020-02-06
WO2018183429A1 (fr) 2018-10-04

Similar Documents

Publication Publication Date Title
US11268071B2 (en) Addition of nucleases directly to cell culture to facilitate digestion and clearance of host cell nucleic acids
US20200347364A1 (en) Aseptic purification process for viruses
US10363304B2 (en) Purification of herpes virus
Carvalho et al. Efficient filtration strategies for the clarification of influenza virus-like particles derived from insect cells
JP6385559B2 (ja) 細胞培養体からのポリオウイルスの精製プロセス
CA2926225A1 (fr) Procedes de detection et d'elimination des rhabdovirus des lignees cellulaires
US11999975B2 (en) Method for purifying an enveloped virus
Makovitzki et al. Evaluation of a downstream process for the recovery and concentration of a Cell-Culture-Derived rVSV-Spike COVID-19 vaccine candidate
WO2022262206A1 (fr) Procédé de purification et application d'un vecteur rétroviral conforme aux gmp
Porter et al. Human papillomavirus quasivirus production and infection of primary human keratinocytes
Castro et al. Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells
JP2022539511A (ja) ウイルスの生産方法及び採取用溶液組成物
Lothert et al. Upstream and downstream processes for viral Nanoplexes as vaccines
JP2005521417A (ja) アデノウイルスの大規模産生方法及びアデノウイルス種子ストック
CN107201371B (zh) 一种携带去优化m基因和两个g基因的重组狂犬病病毒
US20240033345A1 (en) Method for producing virus
Kröber et al. DNA Depletion by precipitation in the purification of cell culture‐derived influenza vaccines
US20210047626A1 (en) Scalable chromatography process for purification of human cytomegalovirus
Eilts et al. Production of baculovirus and stem cells for baculovirus-mediated gene transfer into human mesenchymal stem cells
KR20210075994A (ko) 복제 결함 바이러스 벡터 및 바이러스의 감염성을 측정하는 방법
CN115948616B (zh) 一种柯萨奇病毒b1型的核酸定量检测方法
US20240035002A1 (en) Method for purifying virus
Sedighikamal et al. High-Titer Recombinant Adenovirus 26 Vector GMP Manufacturing in HEK 293 Cells with a Stirred Single-Use Bioreactor for COVID-19 Vaccination Purposes
Fernandes et al. Streamlining the purification of a clinical-grade oncolytic virus for therapeutic applications
KR20100017593A (ko) 바이러스의 증식을 위한 ２-단계 온도 프로파일

Legal Events

Date Code Title Description
STPP Information on status: patent application and granting procedure in general

Free format text: APPLICATION DISPATCHED FROM PREEXAM, NOT YET DOCKETED

AS Assignment

Owner name: MERCK SHARP & DOHME CORP, NEW JERSEY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LI, YI;WOODLING, MATTHEW;KRISTOPEIT, ADAM;SIGNING DATES FROM 20170922 TO 20171204;REEL/FRAME:056276/0799

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

AS Assignment

Owner name: MERCK SHARP & DOHME LLC, NEW JERSEY

Free format text: MERGER;ASSIGNOR:MERCK SHARP & DOHME CORP.;REEL/FRAME:061102/0145

Effective date: 20220407

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: ADVISORY ACTION MAILED

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION